Angiogenesis, i.e., the formation of new blood vessels from pre-existing ones, involves a complex coordination of endothelial cell proliferation, migration, basement membrane degradation and neovessel organization (Ji et al., 1998, FASEB J. 12:1731-1738). The local, uncontrolled release of angiogenic growth factors and/or alterations of the production of natural angiogenic inhibitors, with a consequent alteration of the angiogenic balance (Hanahan et al, 1996, Cell. 86: 353-64) are responsible for the uncontrolled endothelial cell proliferation that takes place during tumor neovascularization and in angiogenesis-dependent diseases (Folkman, 1995, Nat. Med. 1:27-31).
Numerous natural inducers of angiogenesis have been identified, including members of the vascular endothelial growth factor (VEGF) family, angiopoietins, transforming growth factor-α and -β (TGF-α and -β), platelet-derived growth factors (PDGF), tumor necrosis factor-α (TNF-α), interleukins, chemokines, and the members of the fibroblast growth factor (FGF) family. These potent angiogenic factors are often over-expressed by tumor tissues (Presta, 2005, Cytokine & Growth Factors Reviews. 16: 159-178; Grose, 2005, Cytokine & Growth Factors Reviews. 16: 179-186).
Indeed, FGFs, and more specially FGF2, are over-expressed in numerous human cancer including melanoma (Halaban, 1996, Semin Oncol. 23:673-81; Hanada, 2001, Cancer Res. 61: 5511-5516), leukemia (Krejci et al, 2001 Leukemia. 15:228-37, Bieker et al, 2003, Cancer Res. 63: 7241-7246) renal cancer (Hanada, 2001, Cancer Res. 61: 5511-5516), colon cancer (Tassi, 2006, Cancer Res. 66:1191-1198), ovarian cancer (Whitworth at al, 2005, Clin Cancer Res. 11:4282-4288, Gan et al, 2006, Pharm Res. 23:1324-31), prostate cancer (Aigner et al, 2002 Oncogene, 21:5733-42; Kwabi-Addo et al, 2004, Endocr Relat Cancer. 11:709-24) and lung cancer (Takanami et al, 1996, Pathol Res Pract. 192:1113-20; Volm et al, 1997, Anticancer Res. 17:99-103; Brattstrom et al, 1998, Anticancer Res. 18: 1123-1127). In addition, FGF2 over-expression can be correlated with a chemoresistance in certains cancers including bladder, breast, head and neck cancers (Gan et al, 2006, Pharm Res. 23:1324-31). With respect to FGF family members, as FGFs secreted by tumor cells have affinities for the glycosaminoglycan side-chains of cell surface and matrix proteoglycans, these secreted FGFs are most likely sequestered nearby tumor cells forming FGF reservoirs. This particularity makes FGF addressing a good strategy to direct an active molecule that needs a target molecule stably expressed and easily accessible.
Various antibody-based products are currently used as therapeutic drugs and several monoclonal antibodies (mAbs) are now approved in various therapeutic areas such as oncology, inflammation, infectious disease and cardiovascular disease. These mAbs induce tumor cells killing by multiple mechanisms including recruitment of immune system (Harris, 2004, Lancet Oncol, 5: 292-302). The Fc moiety of mAbs is responsible for these immune-mediated effector functions that include two major mechanisms: Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) and Complement-Dependent Cytotoxicity (CDC). ADCC occurs when an mAb first binds via its antigen-binding site to its target on tumor cells, and then the Fc portion is recognized by specific Fc receptors (FcR) on effector cells (i.e. NK, neutrophiles, macrophages . . . ) that attack the target cell. CDC is a process where a cascade of different complement proteins become activated when an mAb binds to C1q leading to formation of C3b on the surface of antibody-coated tumor cells near the site of complement activation. The presence of C3b controls formation of the C5-C9 membrane attack complex that can insert into the membrane to lyse tumor cells (Sharkey, 2007, CA Cancer J Clin, 56: 226-243). The ability of mAbs to stimulate ADCC depends on their isotype. IgG1 and IgG3 antibodies bind highly to FcRs, while IgG4 and IgG2 antibodies bind weakly. CDC capacitity of mAb also depends on mAb isotype. IgG3 and, to a lesser extent, IgG1 are the most effective isotypes for stimulating the classic complement cascade. IgG2 mAbs are less efficient in activating the complement cascade, whereas IgG4 is unable to do so (Strome, 2007, The Oncologist, 12:1084-1095).
The use of a fusion protein that can have, as antibodies, a dual functionality with a binding part exhibiting a specific targeting and an effector part able to induce the lysis of target cells by recruitment of immune system, is one aspect of these therapeutic strategies. In addition, to be useful in therapy, this molecule would need to have advantageous pharmacokinetic properties PK. The Fc moiety can detectably increase the serum half life of the modified soluble FGF receptor Fc fusion, but there is still a need for fusion protein with a longer serum half life. Finally, if this fusion protein is to be used as a drug, it is necessary that it is produced reliably, efficiently and with appropriate productivity.
Thus there is a need for a fusion protein with ADCC and/or CDC activities targeting FGF for treatment of cancer, metastatic tumors and for reducing tumor growth in a subject, with improved PK features, and which can be produced efficiently.
The applicants have now discovered that soluble fusion proteins between soluble FGF receptor part (binding or targeting moiety) and Fc part (effector function moiety) (sFGFR-Fc) that are modified to have a particular glycan profile have in fact substantially improved biological activities, including ADCC and/or CDC activities, and may thus be used as efficacious anti-angiogenic and anti-tumoral drugs, for the treatment of uncontrolled cell growth or cancer. These modified soluble fusion proteins have advantageous PK properties due to their sialylation rate, and can be produced with appropriate productivity and minimal aggregation because of their glycosylation pattern.